Columns and Methods of Use for Analytical Standards and Compounds

ABSTRACT

This invention employs solid phase extraction media and column methods to apply external and internal standards and compounds. Analytical standard or compounds including PFAS are adsorbed to a solid phase extraction media and are stored indefinitely. The standards or compounds remain stable on the solid phase extraction media without decomposing. The standards or compounds may be removed from the solid phase extraction media with an elution solvent or reagent.

FIELD OF THE INVENTION

This invention employs solid phase extraction media and adsorbedmaterial and methods to apply and recover external and internalstandards and compounds including PFAS compounds.

BACKGROUND OF THE INVENTION

The quantification of analytes in samples involves comparing the amountsof the analytes measured in a sample, to analytes in prepared analytestandard solutions (known concentrations). However, preparing andapplying analytical standard compounds for analytical analysis ofsamples can be dangerous due to toxic exposure to the analytical worker.Indeed, the reason that many analytical tests exist is to measure theamounts of dangerous materials in food, drinking water and other drinks,drugs, and cosmetic products to increase product safety to the consumer.And in fact, the handling of many or most standards requires cautioneven under laboratory conditions.

One example is polycyclic aromatic hydrocarbons (PAHs). PAHs exposure tohumans can occur through numerous different routes, namely water, diet,dermal contact with dust or soil, and inhalation of polluted air. PAHshave been shown to induce lung tumors and skin tumors in animals aftertheir metabolic activation.

Due to established carcinogenetic properties of PAHs, they have beenintensively studied in diet. According to the EU's Scientific Committeeon Food (SCF), 15 PAHs have been recognized by SCF as genotoxiccarcinogens, namely benzo(a)anthracene (BaA), benzo[a]pyrene (B[a]P),chrysene (Ch), benzo(b)fluoranthene (BbF), benzo(j)fluoranthene,benzo(g,h,i)perylene, benzo(k)fluoranthene, cyclopenta(c,d)pyrene,dibenzo(a,e)pyrene, dibenzo(a,h)pyrene, dibenz[a,h]anthracene,dibenzo(a,l)pyrene, dibenzo(a,i)pyrene indeno(1,2,3-cd)pyrene, and5-methylchrysene.

To analyze the presence and amount of these materials in food and drink,standards of these toxic materials must be prepared. Primary stocksolutions of high concentration of the materials must be made, and then,serial dilution secondary working standards are prepared. The standardsare added to samples as a liquid solution of known volume containingknown masses of the PAH. In many cases, a mixture of PAHs standards isused to quantify samples.

Samples are prepared to dissolve any PAH material present. Then, knownamounts of standards are added (spiked) as internal standards to thesample, and the PAHs are captured by solid phase extraction.Non-specific materials covered by extraction are removed by washing.After washing, the PAH material that was present in the sample and thespiked PAH materials are removed by elution and analyzed. In some cases,the elution volume is controlled to recover sample in smaller volumes,thus concentrating the analytes. In some cases, the PAH materials areadded as external standards after the sample PAH materials are recoveredby elution.

The use of concentrated standards offers potential exposure of theanalyst to high concentrations of PAH materials. This is the nature ofmaking primary and secondary standard solutions. High concentrations areneeded so that only a small volume of material can be spiked into thesample at the end. If low concentrations are used, then a larger volumespike would have to be made; thus, potentially changing the sample anddisturbing the analytical process due to matrix changes to the sample.There are many other standards that pose risks when making standardsolutions for analytical analysis including: pesticides, residual drug,solvents, natural compounds, etc. Risk to the worker exists, for allanalytical methods, where standards are prepared and added to the sampleinternally or compared to the sample externally. The standards may beprepared in liquid form using a solvent to dissolve the standard.

Another problem with analytical standards or compounds is that many areunstable to light, oxygen, solvents, or other factors. Standards may beprepared and then must be used immediately before they decompose orchange concentration. For example, many flavonoids are unstable in thepresence of oxygen. Flavonoids are a group of plant metabolites, whichare found in a variety of fruits and vegetables, and are thought toprovide health benefits through cell signaling pathways and antioxidanteffects. It is of interest to measure the amounts in various products,but the standards used to quantify them may easily degrade, makinganalysis difficult. In addition, standards may be available in onlylimited quantities and may be expensive. It may be expensive for usersto purchase conventional amounts of standards for preparation of stocksolutions and working standards. In some cases, standards are ubiquitousin the environment and are difficult to prepare.

Per- and polyfluoroalkyl substances (PFAS) are a group of chemicals usedto make fluoropolymer coatings and products. They are now foundeverywhere in the environment and various products and materials.Because PFAS are ubiquitous it is difficult to prepare standards or evenprepare samples to be tested because it can be unknown if the chemicalsand materials used to contain the standards and/or samples are notcontaminated with PFAS materials.

These and other deficiencies in the art are remedied by the claimedinvention and embodiments described below.

SUMMARY OF THE INVENTION

Embodiments of this invention employ solid phase extraction media andmethods to apply external and internal standards or compounds foranalytical analysis or other uses. In some embodiments, the media iscontained within a column. In these embodiments, the columns can bepipette tip columns or other types of columns that can supportbidirectional flow of buffer or solvents through the column. In someembodiments, the columns are pipette tip columns, spin columns, plates,pressure flow columns and vacuum flow columns that supportunidirectional flow. The columns contain solid phase extraction resinmaterials that are suitable for adsorbing the particular type ofanalytical standard(s), compounds, or samples.

In other embodiments, the solid phase extraction media is magneticbeads. In other embodiments, the solid phase extraction media is incolumns. In other embodiments, the solid phase extraction media iscontained within a vial or disc.

Standards or compounds are adsorbed and taken up from a solvent andstored on the solid phase extraction column. The term, “solvent” as usedherein is a liquid. It can be organic or aqueous or a mixture of organicand aqueous. The stored standards or compounds on solid phase extractionmedia to not contain elution reagents. Elution reagents may be solvent,buffers, acids, bases or other reagents.

In some embodiments, the solvent is removed and standards are stored onthe dry resin within a column. The standards or compounds may be storedindefinitely. The standards or compounds may be stored on the solidphase material for certain specified times or shelf life. The stabilityof a compound adsorbed and dried onto a solid phase extraction surfacemay be a day or greater, a week or greater, a month or greater, a yearor greater, or two more years. Stability is defined as the compound isstored intact on the solid phase surface without residual solvent orelution reagents and then recovered by the addition of solvent orelution reagents. The chemical nature under which the materials arestored on the solid phase material is adsorption onto the functionalgroups of the solid phase extraction media.

Materials are adsorbed to the resin solid phase extraction surface andthen dried to remove the solvent. Storage of the compound is on thesurface without any residual solvent or elution reagent so that thedeposition of the compound is directly on the solid phase materialwithout any solvent stabilizing the compound or shielding the surface ofthe compound. Because of this, the compound conforms to the surface andmolecules of the solid phase material. Surprisingly, standards,compounds, and samples stored in this manner were found to offerenhanced stability.

More surprisingly, the compounds or materials are successfully desorbedfrom the solid phase extraction material under conditions of desorptionfor compounds from solution. Normally, compounds that are adsorbed fromsamples by traditional solid phase extraction have solvent moleculesassociated with the adsorbed sample compound and with the solid phasemedia functional group. In desorption, these solvent molecules arethought to work in tandem with the desorption solvent molecules to liftthe compound from the solid phase extraction surface. In the compoundsof the invention, the compound and surface have much fewer or even zeroelution solvent molecules when adsorbed to a solid phase extractionsurface. Compounds and the solid phase surface that have had solventmolecules removed are not available to work in tandem with elutionsolvent molecules to assist in desorption.

The amount of standard on the stored column or solid phase extractionmedia is generally of sufficient quantity to compare to a single samplecontaining (unknown) analytes. Thus, the mass of the standard containedon the column is many times lower than the mass contained in a typicalprimary standard or secondary standard solution. Columns containing thelower amounts of materials are much less likely to be dangerous for theworker to handle. Solid phase extraction media in a column havingadsorbed standards may be called an on-column standard. One or morestandards in parallel may be adsorbed under the same and predictableconditions to solid phase extraction materials. It should be noted thatin this procedure, the elution solvent, or buffer or reagent moleculesassociated with the compounds are removed in the process. The process isfree of elution solvent, buffers or reagents.

In some embodiments, standards or compounds may be stored in solventfree adsorption in larger quantities to be later be desorbed and used toprepare standard solutions or used for other purposes. In someembodiments, the material stored on the media or in columns may be usedfor other purposes besides analytical work.

In some embodiments, solvent free adsorbed compounds on solid phaseextraction material may be contained in columns that are shielded fromlight. One or more columns may be contained in a box that is lightproof. One or more columns may be contained in a sleeve or pouch thatdoes not transmit light and shields the columns.

For example, many drugs are sensitive to light and therefore theirformulated products may degrade during manufacturing, storage, andadministration. This may result in potency loss, altered efficacy, andadverse biological effects and therefore, analytical quantification mustbe performed periodically on stored drugs. Standards on solid phaseextraction media could be stored in a container or pouch that preventedlight from reaching the compound. Interestingly, since the standard maybe stored internally in the solid phase extraction media, protectionfrom light is afforded the standards even without the use of an opaquecovering or pouch.

In some embodiments, on-column standards or media are stored with opaquecolumn or covering, shielding the column from light.

In some embodiments, on-column standards or media are stored undervacuum i.e., vacuum-packed. Vacuum may be maintained indefinitely. Insome embodiments, cooling may be applied to the material along withvacuum to provide a stable standard or compounds. In some embodiments,heat may be applied to the material along with vacuum to provide astable standard or compounds. In some embodiments, on-column standardsor media are stored under inert gases such as nitrogen, helium, or othernoble gases.

Any type of protective covering, temperature, or gas may be used toprotect the compounds adsorbed on the solid phase extraction columns ormedia from degrading. In some embodiments, the on-column standards ormedia are stored at temperatures lower than ambient or room temperatureto protect the compounds adsorbed on the solid phase extraction resin ormedia. In some embodiments, the on-column standards or media are keptcold for long term storage.

In some embodiments of the invention, materials or compounds may bestored on solid phase media. The adsorbed materials or compounds may bedesorbed from the columns or media and used as external standards toquantify sample analytes. In some embodiments, sample compounds or othercompounds may be stored on media and then recovered.

Columns containing known different amounts of internal standards providedata for standard addition analysis of analytes; thus, eliminatingmatrix effects of the sample matrix (e.g., ion suppression).

By using a column format with solid phase media to contain the toxic andcarcinogenic standard materials, exposure to the worker is reduceddramatically, perhaps a thousand to a million times or more. The exactreduction of risk depends on the mass amount of a primary standardcompared to the number of on-column standards employed in a singleanalytical operation. Risk is also lowered by limiting the operations.Thus, human risk is lowered by limiting the exposure to large amounts ofstandards and by limiting the manipulation of standards. In addition,having the option of employing internal standard methodology eliminatesthe need to pipette or handle primary or secondary standard solutions.Finally, the entire process can be automated in a high-throughput SPEautomated workflow reducing possible exposure of toxic and carcinogenicmaterials to the worker and eliminating the risk of human and phenotypicmutations.

In some embodiments of the invention, solid phase media is in the formof beads including magnetic beads or in the form of fibers. In someembodiments of the invention, solid phase media may be in vials, columnsor discs.

Embodiments of the invention have the advantage of limiting the exposureof the analytical worker to toxic materials and lowering theconcentration and amount of standards needed to perform analysis.Embodiments of the invention have the advantage of providing standardsin a format that limits exposure of the standard compound or compoundsto degrading conditions, such as: light, air, or solvents, etc., whichis useful for standards or compounds that are unstable in air, light orsolvent. Embodiments of the invention have the advantage of reducing theamounts, and thus reducing waste, of standards or compounds that areprecious or expensive. Embodiments of the invention have the advantageof limiting hands-on work and facilitating the automation of the use ofstandards in the analytical process, thereby improving productivity andreducing waste.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Adsorbing standards onto a solid phase extraction resin phaseby evaporation of solvent.

FIG. 2 . Adsorbing standard onto columns through back-and-forth flow ofa standard solution through the solid phase extraction column bed. Thesolvent is removed after adsorption.

FIG. 3 . Adsorbing standard on a flow through type solid phaseextraction column by adsorption of a standard solution onto the columnresin. The solvent is removed after adsorption.

FIG. 4 . Adsorbing standard on a column by evaporation removal of thesolvent from a standard solution onto the solid phase extraction columnresin.

FIG. 5 . HPLC-FLD chromatograms of contaminated Primulae flos extract:

Chromatogram A) SPE pipette tip column without standards adsorbed;Chromatogram B) on-column standard SPE pipette tip column, Peak 1:B(a)A, Peak 2: Ch. Chromatogram A and B are used to calculate theconcentration based from the amount of standards preloaded onto thepipette tip column.

DETAILED DESCRIPTION OF THE INVENTION

The quantification of trace compounds in high matrix samples is often achallenge in analytical chemistry. This is especially true when specificcompounds or compound classes are regulated by official authorities dueto their toxicity, carcinogenicity or endocrine activity, e.g.,parabens. To quantify analytes, standard solutions are prepared assolutions and then added during the sample analysis, either externallyor internally. Standard solutions are prepared as a primary standard orstandard mixture at relatively high concentrations, and secondarystandard solutions are used in the analytical process, prepared bydilution of primary standards.

In embodiments of this invention, standards, compounds, or samples areadsorbed onto an extraction resin phase which can be contained in apipette tip column, flow through column, plate or any other columnformat. The column formats may employ unidirectional or bidirectionalflow.

One or more standards or compounds may be adsorbed to the media whichcan be disposed within a column. Different standards may have differentconcentrations loaded to enable an analysis of analytes present atdifferent concentrations. The standards are dried and stored untilneeded. The standards may be stored under vacuum, under cool conditions,the absence of light, in the presence of an inert gas, etc. depending onthe requirements analyte on the column to prevent degradation. However,surprisingly, the removal of the solvent from the column afteradsorption can be performed and stability of the compound is maintained.Surprisingly, the stability of analytes, samples or compounds isincreased after they have been adsorbed onto the solid phase materials.

Furthermore, the re-introduction of a solvent may, depending on thesolvent, release the compound bound to the solvent phase extractionsurface. Complete release is defined as greater than 80% of the originalcompound, greater than 85%, 90% or 95% of the original compound massamount. This is surprising, because the adsorbed molecule is maintainedunder completely dry conditions, meaning the compound molecules are inintimate contact with the solid phase extraction media in the absence ofsolvent molecules. This means that the analyte or compound is inintimate contact with the solid phase material through bonding thatwould not be present if elution solvent or molecules were present. Yetthe compound can be completely taken up by the fresh application ofelution solvent or molecules and released from the solid phaseextraction surface. The elution solvent or molecules may be comprised ofan organic and/or aqueous liquid phase and may contain salts, organics,acid, base or buffer.

Standards are adsorbed, taken up from a solvent, and stored on the solidphase extraction media. The media can be disposed within a column. Insome embodiments, the solvent is removed, and the standards are storedas dry resin within the column or on the resin. In some embodiments, thestandards on the column are free from any liquid.

In other embodiments, liquid can be present provided the liquid does notcontain elution reagents including, acids, bases or buffers capable ofelution. For example, PFAS standards adsorbed onto ion exchange solidphase extraction media, may not contain elution reagents. Elutionreagents are defined herein as any substance capable of displacing thestandards.

The standards or compounds of the invention may be stored indefinitely.The standards or compounds may be stored on the solid phase material forcertain specified times or shelf life. The stability is determined bythe nature under which the materials are stored on the solid phasematerial. Materials are adsorbed to the resin solid phase extractionsurface and then can be dried or treated to remove the solvent orelution reagent.

In some embodiments, the column is substantially free of solvent. Insome embodiments, the column can be free of eluting reagents. The amountof solvent left can be described as 100% solid phase extraction materialmass and 0% solvent mass, 99% solid phase extraction material and 1%solvent mass, 98% solid phase extraction material and 2% solvent mass,97% solid phase extraction material and 3% solvent mass, 96% solid phaseextraction material and 4% solvent mass, or 95% solid phase extractionmaterial and 5% solvent mass. In all these embodiments, the column issubstantially free of solvent. The amount of solvent remaining afterdrying can be less than 5%, less than 4%, less than 3%, less than 2%, orless than 1% of the original solvent or compound solvent matrix.

Standards, compounds, and samples stored in this manner were found to bestable indefinitely. Stable is defined as greater than 80, 90, 95, 96,97, 98 or 99% of the original compound remains. The stability time isdefined as greater than 1, 2, 3, 4, 5, 6, or 7 days. The stability timeis defined as greater than 1, 2, 3, 4, 10, 20 30, 40, 50, or 100 weeks.

The standard or materials being stored may be inorganic or organic. Theymay be ions, metals, or inorganic or organic compounds. They may bebiomolecules including smaller organic compounds or larger biomoleculessuch as peptides, proteins, lipids, ssDNA and dsDNA, nucleic acids andcomplex organic biomolecules.

The solid phase material may be inorganic or organic. It may be an ionexchanger, reverse phase, normal phase or hydrophobic interaction. Itmay contain silica, zirconia, and alumina, titanium oxide,hydroxyapatite, etc. It may be non-porous polymer, macroporous polymer,glass, controlled pore glass, carbon, activated carbon, ceramic, etc. Itmay be polystyrene/divinylbenzene, methacrylate ester, olefin polymer,etc. In some embodiments of the invention, any material may be used thatcan act as a solid phase extraction material that can adsorb compoundsand that can release compounds.

Preparing the Adsorbed Solid Phase Extraction Media Standards

Preparing the standards may be performed in several different ways. FIG.1 shows how a standard can be dissolved in solution then mixed with asolid phase extraction resin. The mixture is rotated in a round bottomflask, and vacuum and/or heat is applied to the flask evaporating thesolvent and adsorbing the standard(s) to the resin. Any type of mixing,including stirring, may be performed to allow uniform adsorption to theresin or media. The solvent may be of any type to allow dissolving ofthe standard and eventual evaporation. Optional heat may be applied withvacuum to enhance or speed solvent evaporation. After coating, the resinmay be packed into a column and will be ready for use. The amount ofresin packed into the column may be specified.

The standard may be adsorbed directly onto solid phase extraction media.In some embodiments, the media is contained in a packed bed column. InFIG. 2 , the solution containing the standards is passed back and forththrough the column. For adsorption from a solvent, the selectivity ofthe standard for the resin is high and adsorbed directly. An example ofthis process is passing PAH standards dissolved in methanol/waterthrough a PS/DVB solid phase extraction column. The column has a muchhigher selectivity for the PAH standard than the methanol/water liquidphase. After adsorption, the resin is dried by standing or optional airflow and/or heat, and the on-column standard is ready for use.

The column depicted in FIG. 2 is a back-and-forth flow column. In FIG. 3, a flow through or unidirectional column is shown with a coatingprocedure similar to FIG. 2 . The standard is completely adsorbed, or aspecific, known amount is adsorbed by the resin from the solvent.

In FIG. 4 , another method is shown where the solvent containing thecompounds covers the column resin, and the solvent is removed. In someembodiments, standards are on an extraction medium within a packed bedcolumn. The column may be dual flow, as shown in the figure, orunidirectional flow. The standard(s) or compound(s) containing solutionis loaded into a column just covering the resin bed. The solvent isevaporated in the column leaving the standard adsorbed to the resin bed.Evaporation can be done slowly at room temperature, or the column may beheated in an oven, evaporating solvent away more quickly.

In some embodiments, the amount of standard or compounds captured on thecolumn is known. In some embodiments, the number of compounds capturedon the column is not known.

Using the Standards

The standards may also be used as a source for external standards. Inthis case, the standards are desorbed from the media or column bed in asmall volume of buffer or solvent and quantified alongside samplesolutions as part of an analytical method. The amount of materialreleased from the solid phase and solvent volume is known; thus, theconcentration of the standard can be calculated and is known.

The analyte is quantified by comparing the known amount of the standardsolution prepared from the standard to the unknown amount of analyte.This procedure depends on being able to prepare without weighing astandard solution of known concentration from the standard. There areseveral advantages to this method. These include only preparing thestandard solution that is needed to process the samples and using onlysmall amounts of standard in terms of mass amount and volume amount.

Another, more elegant and less error-prone method is using the on-columnstandards as internal standards. In this embodiment, the on-columnstandard columns containing the standards are also used as solid phaseextraction columns to capture analytes from the samples. This method canalso be used with standards adsorbed to solid phase extraction mediawhere the media is not contained in a column.

Solid phase extraction normally consists of three steps. Capture of theanalyte from a sample, washing away and removal of any contaminants, andthen elution and collection of the analyte. An internal standard may beadded to the sample to mimic the conditions of the standard to thesample analyte. In this way, any increase in detection signal is due tothe internal standard that has undergone the same process and conditionsas the sample analyte. However, in the columns and method of theinvention, the standard is already present on the column. The adsorptionconditions of the standard and the sample analyte are different.Nevertheless, after capture and removal of contaminants, standards andanalytes may be eluted together. The on-column standards may serve asinternal standards to quantify the analytes. Columns containing knowndifferent amounts of internal standards may provide data to allowstandard addition analysis of analytes. The on-column standards of theinvention may have a dual function and may also be used as solid phaseextraction columns to capture analytes. As the columns are being used tocapture sample analytes, they contribute various and known amounts ofinternal standards to the samples. Standard addition curves areconstructed to calculate the original (starting) concentration of theanalyte in the sample. Samples analyzed using internal standards areindependent from matrix effects such as mass spectrometry ionsuppression.

It is important to note that the operation of on-column standardscontaining the standards is different from the operation of solid phaseextraction columns (sample columns). On-column standards are meant toprovide standard analytes to the sample. Sample extraction columns aremeant to adsorb analytes from the sample and then later release sampleanalytes for analysis. The released samples are measured either usingexternal standards or internal standards, both of which can be providedby the on-column standards.

It is also important to note that bidirectional flow allows theinteraction of the standards and analytes to come to equilibrium wheresome of the material may be adsorbed onto the column, and some may bedesorbed into solution. Since the actions are the opposite of on-columnstandards and analytes, it is important for both standards and sampleanalytes to come to the same equilibrium.

The process is based on a back-and-forth flow process where materialsare brought into equilibrium under a given set of conditions. Forexample, pipette columns with solid phase extraction media are placed ona pipette pump or a liquid handling robotic pump. The pipette tipcolumns are placed in the sample containing the sample analyte. Liquidflows through the pipette tip column using back and forth flow. Oncolumn standards may be (perhaps partially) desorbed from the solidphase medial while sample analytes of the same identity as the standardsare being adsorbed. The process may be complicated because standards arebeing released while analytes are being captured. The technology isbased on capturing sample materials and also releasing standardmaterials from the tip columns.

The internal standard process allows the on-column adsorbed standard andsample analyte to come to equilibrium under identical matrix conditions.After capture and removal of contaminants, standards and analytes areeluted together.

Unidirectional columns may also be used for on-column standards. Theonly requirement is having data on the extent of capture and release ofstandards or compounds from the solid phase extraction columns. Mostuseful are conditions where the capture of the recovery of the analyteor compounds is complete from both the on-column standard column andfrom the solid phase extraction column.

Safety to the analytical worker is dramatically improved using on-columnstandards due to much lower amounts of toxic materials used in theanalysis. Safety is improved further by automating the use of thecolumns. One objective of the columns of the invention was to establisha high-throughput SPE-automated workflow with minimized personalexposure to tremendously carcinogenic compounds.

Standards of the invention can accurately determine the lowconcentrations of toxins or carcinogens in complex matrices whileminimizing worker exposure of the toxins or carcinogens during thehandling of samples.

Due to the high relevance, existing legislative regulations, availablestandard compounds, and methodologies for separation and detection,polycyclic aromatic hydrocarbons (PAHs) on-column standards were chosenfor an experiment. In one example, polystyrene-divinyl benzene (PS/DVB)macroporous resin 80 μL bed based on-column standards, containing PAHsat different concentration levels, were developed for quantification ofPAHs in plant extracts. High adsorption selectivity of PAHs, on thePS/DVB sorbent from methanol, permitted the design of SPE on-columncalibration-set of 50, 150, 250, 350, 450, 500, 1500, 2500, 3500, and4500 ng of the standard mixture of total PAH mass on the pipette tipcolumns. Elution of the standards (and sample analytes) from the columnwas shown to be possible with 100% tetrahydrofuran (THF) solvent.

In some embodiments of the invention, analytical standards of theinvention are PFAS standard compounds. Per-and polyfluoroalkylsubstances (PFAS) are a group of chemicals used to make fluoropolymercoatings and products that resist heat, oil, stains, grease, and water.Fluoropolymer coatings can be found in a variety of products. PFAS areman-made chemicals that include perfluorooctanesulfonic acid (PFOS) andmany other chemicals. Other PFAS include, again only as examples,perfluorooctanoic acid (PFOA, perfluorononanoic acid (PFNA) andperfluoroalkyl acids (PFAAs). PFAS can be found in any type of waterincluding drinking water, rain water, lake, river and ocean waters. Theycan be found in virtually any kind of manufactured and natural productsor substances including, only as examples, textiles, leather, paper andpaints, fire-fighting foams, and wire insulation. PFAS can be found inin any water or solvent in contact with these products or substances.PFAS can also be found in biological tissue and fluids. Methods usingsolid phase extraction media of the invention can be used to measurePFAS in all natural and man-made substances or sample and in biologicalsamples.

In some embodiments of the invention, the standards are fully loaded onthe column from the standard solution to prepare the on-columnstandards. In this way, the mass amount of standard brought to theanalysis is known.

In some embodiments of the invention, if the on-column standards areused in an internal standard mode, the standards remain on the mediawithin the column during the column activation process. The activationsolvent activates or wets the resin without removing the standards fromthe column.

In some embodiments of the invention, the on-column standard is notactivated prior to sample capture. In some embodiments of the invention,the resin containing the standard on the on-column standard is waterwettable and does not require solvent activation.

In some embodiments of the invention, the optionally activated on-columnstandard is used to capture compounds from a sample. The standards heldon the column and analytes in the sample come to equilibrium under themobile phase and stationary phase conditions. During the captureprocess, in some embodiments, the standards remain completely adsorbedon the column. In some embodiments, the standards remain mostly adsorbedto the column and are slightly released from the resin or slightlydesorbed. During the capture process, in some embodiments, the sampleanalytes are completely taken up and adsorbed on the column. Theequilibrium of standards and analytes are the same, but in some cases,standards are slightly released from the column and/or sample analytesare not fully taken up by the column.

In some embodiments of the invention, the on-column standard containingstandards and captured sample analytes are eluted and recovered.Analytes are analyzed using standard addition methodology.

The solid phase media of the invention may be in any form includingmagnetic beads, beads and, fiber. It can be in any format includingcolumns and discs. Sample processing may be automated in a fashioncompatible with the solid phase media.

An example of a robotic system was used to automate the process. APhyNexus® (San Jose, Calif.) MEA™ Purification System was used for theautomation of the PAH solid phase extraction (SPE) method. The MEA™System is a 12-channel liquid handling system equipped with a fullyprogrammable positioning of the 12-channel pipette. It has the abilityto place sample extraction pipette tip columns, washing solutions andelution plates in any available tray position. The activation, loading,washing, and elution solutions were placed in Abgene™ 96 well 2.2 mLpolypropylene deep well U-shaped storage plates (occupying one trayposition out of the available eight). The software enabled theprogramming of SPE method for managing the entire SPE procedure fromactivation to elution. Table 1 shows the major steps of the automatedSPE method.

TABLE 1 PAH extraction method parameters for MEA ™ Purification SystemTray Position Function Protocol Specification (1000+) 1.1 HomeLoad-columns 3.1 SPE-Activation Down 2400, cycle 500 μL 5 times 3.2SPE-Loading Down 2635, cycle 800 μL 10 times 5 sec delay 3.3 SPE-WashingDown 2400, cycle 500 μL 10 times 5 sec delay 1.1 Column Partial Down2600, eject, up, intake 1200 μL, Drying load tips, expel 1200 μL Down2600, eject, up, intake 1200 μL, load tips, expel 1200 μL Down 2600,eject, up, intake 1200 μL, load tips, expel 1200 μL 3.4 SPE-Elution 1Intake 700 μL, down 2635 cycle 500 μL 10 times, 5 sec delay 3.5SPE-Elution 2 Intake 700 μL, down 2635 cycle 500 μL 10 times, 5 secdelay 3.6 SPE-Elution 3 Intake 700 μL, down 2635 cycle 500 μL 10 times,5 sec delay 1.1 Home Down 2600, Eject columns

The complete SPE method from activation to elution was executed in 17minutes for a single row of 12 samples. Highly complex samples such asplant extracts, food extracts, beverages, etc. are adsorbed byback-and-forth flow enabling equilibrium between stationary and mobilephase on the SPE material in the pipette tip column.

Column washing to remove contaminants from the pipette tip column isperformed by back-and-forth flow enabling equilibrium between stationaryand mobile phase to remove matrix compounds. Finally, elution ofcompounds of interest by back-and-forth flow produces an equilibriumbetween the stationary and the eluent mobile phases to recover compoundof interest and the internal standards. Quantification by LC-MS or GS-MSdata is performed by the standard addition method.

Similar procedures would be used in other robotic liquid handlers. Theactivation step is optional; however, if an activation step is includedin an internal standard procedure, the activation solvent optimallyshould not remove the standards from the column. If standards areremoved, then they must be added to the sample. This dilutes theactivation solvent and standards, and a sample can be captured under thesame matrix and solvent conditions. Another method is to use a resinthat is water wettable but still has non-polar selectivity. In this way,activation of the column may not be necessary. Finally, as Table 1shows, elution may be performed with one or more aliquots. If more thanone aliquot is used, then the volumes can be combined at the end of theprocedure.

The stability of flavonoids myricetin, quercetin, and kaempferol boundto SPE sorbents as on-columns standards were studied. These compoundswere selected for the evaluation because flavonoids can be unstable anddegrade when exposed to light and oxygen. Flavonoids are one of thelargest groups of plant secondary metabolites. Flavonoids are importantfor stress response in plants, act as pigment sources for flowercoloring, and play an important role in interaction with insects. Forhumans and animals, they are present in food, act as antioxidants, andhave estrogenic effects. They are antimicrobial and anti-cancerous.Quercetin, kaempferol, and myricetin can protect human skin from UVlight.

This work describes the effectiveness standards, samples or compoundsstabilized on solid phase media compared to the wet (solvent) storage.Storage of flavonoids in solution degrade due to atmospheric oxygendissolved in the solution.

Stock solutions of kaemferol, quercetin, and myricetin were prepared bydissolving 5 mg of each compound in 2 mL methanol. All three solutionswere combined to a final volume of 10.0 mL using pure methanol. Fromthis stock solution, working standards at net final concentration of10-30 ppm (each) were prepared by appropriate dilution of the stocksolution into 1:1 methanol/water, v/v. All working solutions were storedat −20° C. as well as stored at room temperature (stability wasmonitored after 4 hours, 1 day, 3 days, 7 days, 10 days and 28 days'storage at room temperature) to monitor the extent of degradation.

In addition, on-column standards were prepared from the fresh standards.Unidirectional flow Oasis® HLB, 30 mg sorbent 1 mL cartridges were usedas the solid phase extraction column. The appropriate amounts ofstandard compounds corresponding to 10-30 ppm were adsorbed to SPEsorbent Oasis® HLB cartridges.

The SPE columns were activated with 1 mL methanol and equilibrated with1 mL water. The cartridges loaded with standard compound solutions driedand were stored in vacuum sealed packing.

The columns were stored for several days. The columns were washed with1:1 methanol/water. Elution was performed twice with 500 μL 10% formicacid in methanol. HPLC-UV and ESI-MS were used to quantify the targetcompounds.

Degradation Kinetics (0-28 Days) for wet storage at room temperature.

For the wet storage of standard compounds at room temperature, myricetin(six hydroxyl groups) showed maximum instability among the testedcompounds, followed by quercetin (five hydroxyl groups) and kaempferol(four hydroxyl groups). Myricetin demonstrated a 17-30% degradation forthe standards (10-30 ppm) over a period of 28 days with the averagedegradation accounting for 24.35%±6.6 (average±SD), while quercetindisplayed 5-12% degradation (8.34%±2.87) for 10-30 ppm standards.Instability of less than 5% (2.69%±1.58) for kaempferol was observed.Tables 2, 3, and 4 show the extent of the degradation of testedcompounds.

TABLE 2 Degradation kinetics for myricetin for the standards stored insolution form at room temperature (percent degradation) 0 4 1 3 7 10 28Myricetin Hrs Hrs day Day Day day Day 10 ppm 0 −0.4796 −0.9592 4.55611.27 12.47 30.46 15 ppm 0 −0.6270 0.3134 5.015 9.404 10.66 31.97 20 ppm0 1.520 2.690 6.901 12.74 12.63 22.92 25 ppm 0 0.1736 3.211 5.990 10.679.982 19.36 30 ppm 0 0.4265 0.9239 3.767 8.319 7.747 17.13

TABLE 3 Degradation kinetics for Quercetin for the standards stored insolution form at room temperature (percent degradation) 0 4 1 3 7 10 28Quercetin Hrs Hrs day Day Day day Day 10 ppm 0 −0.4024 0.4024 3.8226.237 5.432 12.47 15 ppm 0 0.1356 −0.4070 2.442 3.799 3.664 9.633 20 ppm0 −0.4094 0.1023 1.842 4.811 3.480 7.881 25 ppm 0 0.4552 1.442 2.6564.780 3.414 6.525 30 ppm 0 −0.3121 −0.2496 0.9363 3.246 2.871 5.056

TABLE 4 Degradation kinetics for kaempferol for the standards stored insolution form at room temperature (percent degradation) 0 4 1 3 7 10 28Kaempferol Hrs Hrs day Day Day day day 10 ppm 0 −2.304 0.000 0.70921.773 0.5319 4.610 15 ppm 0 −1.430 −0.9535 0.2384 1.430 0.3576 3.933 20ppm 0 −1.736 −1.188 −0.5484 0.8227 -2.285 0.8227 25 ppm 0 0.4729 0.87841.149 2.635 0.000 1.554 30 ppm 0 −0.5039 −1.064 −0.4479 1.063 0.78392.580

The recovery of 10, 15, and 20 ppm standard solutions from freshlyprepared Oasis SPE cartridges are described in Table 5.

TABLE 5 SPE recovery data for standards Bound % (Oasis ® HLB, Standard30 mg, 1 ml sorbent) Recovery % Myricetin 100  95-102% Quercetin 100102-105% Kaempferol 100 102-105%

Comparison of the Stability at Different Storage Methods.

The stability of flavonoids was compared at wet storage (solution format room temperature and at −20° C.) and dry storage (room temperaturebound to sorbent and dry form in an open Eppendorf vial). The wetstorage at room temperature demonstrated a continuous degradationpattern for the investigated compounds. The phenomenon was pronouncedfor myricetin which revealed an average recovery of 75.84%±6.5 after 28days of wet storage. This is followed by quercetin with 91.2%±3.79recovery and kaemferol with 96.5%±1.58 recovery rates. When the samestandards were evaluated for the on-sorbent storage at room temperature,a degradation trend was not observed. Quercetin and kaempferol wererecovered 103%±4.06 and 105%±4.80, respectively. Myricetin was recovered88.9%±2.5. For myricetin, loss of the recovery can be attributed to SPErecovery rates of this compound (95% optimum recovery). The wet storageat −20° C. and the open vial dry form storage did not show anydegradation pattern and could achieve the recovery rates of 100-110%.

TABLE 6 recovery of Myricetin standards (10-30 ppm) at different storagetechniques after 28 days Myricetin Recovery Rates 28 Days Conc. 0 HrsWet storage Wet Storage (ppm) Value (−20 C.) (Room Temp.) 10 100 108.5470.73 15 100 104.08 68.03 20 100 103.04 77.08 25 100 101.65 80.64 30 10099.57 82.87

TABLE 7 Recovery of Quercetin standards (10-30 ppm) from differentstorage techniques after 28 days Quercetin Recovery Rates 28 Days Conc.0 Hrs Wet storage Wet Storage (ppm) Value (−20 C.) (Room Temp.) 10 100101.00 86.65 15 100 101.62 88.92 20 100 101.63 90.63 25 100 100.00 93.4730 100 98.56 96.33

TABLE 8 Recovery of Kaempferol standards (10-30 ppm) from differentstorage techniques after 28 days Kaempferol Recovery Rates 28 Days Conc.0 Hrs Wet storage Wet Storage (ppm) Value (−20 C.) (Room Temp.) 10 100101.06 95.39 15 100 100.95 96.07 20 100 102.74 99.18 25 100 100.54 98.4530 100 99.89 97.48

Tables 7 and 8 show that Quercetin and Kaempferol were recovered at 103%and 105% respectively, after 28 days bound to the sorbent. Table 6 showsthat on-column Myricetin was recovered at around 90% after 28 daysstorage at room temperature bound on the sorbent. The results arecomparable to wet storage at −20 degrees C.

Many solid phase extraction material types may be used to storeon-column or on-media standards or compounds. The media may beinorganic, mineral, organic or polymeric resins. The media or resins maybe macroporous or microporous. The compounds may be stored on the mediaor resin surface or internally. The compounds may be drugs,environmental pollutants, contaminants. The samples may be water, plantextracts, body fluids, etc.

The on-column standards may be used to quantify the screening of toxicsubstances from any sample matrix. The preloaded sorbent approach canprovide rapid screening for the hazardous substances from any herbal,cosmetic or biological fluids.

Example 1. Preparation of PAH On-Column Standards

Primary stock solutions of PAHs, namely B(a)A, Ch, B(b)F and B(a)P, wereprepared by dissolving 1-2 mg of each compound in 10.0 mL methanolseparately. From these stock solutions, secondary working standard stocksolutions were mixed together to constitute the working standardscontaining all the standards mixed together. The secondary standardmixtures were prepared to have final concentrations of 0.1-10 ppm. Allprimary and secondary working solutions were stored at −20° C.

PS-DVB 160 μL resin bed volume pipette tip columns (50 mg resin,particle size 70-100 μm particle size, 950 m²/g surface area) fromPhyNexus® (San Jose, Calif.) were activated with 500 μL of 1:1methanol/water through five times recycling of the activation solution.The loading was achieved through ten times recycling of the 500 μLsample. Washing was carried out once, with 1:1 methanol/water (ten timesrecycling of 500 μL wash solution). After washing, the sorbent was driedfor 1 minute through flushing ambient air in and out of the tip and thenair dried and stored for 15 days.

PS-DVB sorbent retains PAHs due to strong π-π interactions betweensorbent and PAHs. The PS/DVB on-column standards were prepared byloading 0.1, 0.3, 0.5, 0.7, 0.9, 1, 3, 5, 7, and 9 ppm standardmixtures. These corresponded to 50, 150, 250, 350, 450, 500, 1500, 2500,3500, and 4500 ng by weight of each of the four PAH standards containedin the secondary standard mixture.

Table 9 represents the bound percent and recovery data for PAH standardsfrom on-column standards. Loading capacity of 50 mg PS-DVB columns werefound to be more than 95% for the investigated concentration range from0.1 to 9 ppm. The recovery of standards was in the range from 90-102%(relative standard deviation <8%) showing that the on-columns standardsmay be stored and used later.

TABLE 9 Bound and recovery data of PAH standards after 15 days ofstorage of on-column standards. Average % Average % Standards 0.1-9 ppmBound Recovered Benz(a)anthracene (B(a)A) 95-99 100-102 Chrysene (Ch)95-99 100-102 Benzo(b)fluoranthene (B(b)F) 95-99 90-97 Benzo(a)pyrene(B(a)P) 95-99  96-100

Example 2. Quantification of PAHs of an Extract from ContaminatedPrimulae flos, an Anti-Viral and Anti-Inflammatory Herbal Medicine

Five grams of contaminated Primulae flos was extracted with 50 mL ofethyl acetate: cyclohexane (1:1, v/v) through ultra-sonication for 30minutes. The extract was filtered, dried under nitrogen stream, andreconstituted in 5 mL of methanol. Using automation, 500 μL of thesample was loaded onto a PS-DVB pipette tip column, without internalstandards, and 500 μL each of the sample was loaded onto 3 differentinternal standard columns, i.e., 0.3, 0.5 and 0.7 ppm benz(a)anthraceneand chrysene PS-DVB on-column standards. The internal standard SPE wasperformed as described in Table 1. The elution volumes were combined andanalyzed by HPLC-FLD analysis with excitation at 270 nm and detectionemission 390 nm.

FIG. 5 shows HPLC-FLD chromatograms of contaminated Primulae flosextract. Chromatogram A is the extraction without using an on-columnstandard. Chromatogram B is the chromatography obtained with an 0.5 ppmon-column standard. Peak 1 is benz(a)anthracene and peak 2 is Chrysene.The concentrations of the two PAH were calculated using an internalstandard plot generated using the peak heights. Benz(a)anthracene wasdetermined to be present at 60.9 μg/Kg Primulae flos and chrysene wasdetermined to be 70.8 ug/Kg Primulae flos.

Example 3. Quantification of THC in CBD-Extracts

Due to its beneficial properties (i.e., skin calming), CBD extracts areused in nutrition products and cosmetics. In several countries,limitations of THC-content in CBD extracts exist (in Europe 0.2%-0.3%).The preloaded column technology of the invention enables laboratorieswithout drug certification to perform quality control. The amount ofpreloaded THC-standard is on the column and may not be regulated.

Example 4. Quantification of Furanocoumarins in Cosmetics Products

Furanocoumarins are a class of photo-toxic compounds, thus their contentin products for topical applications is limited in the European Union.The preloaded columns of the invention provide a safe, quick, andeasy-to-use methodology for determining the furanocoumarine-content incosmetics as well as phyto-medicines like healing-ointments.

Example 5. Quantification of Ginkgolic-Acids

Ginkgo-extracts play an important role in traditional Chinese medicineand can be found in numerous products. Due to the toxicity of ginkgolicacids, their maximum level in phytopharmaceuticals containing ginkgoextracts has been recently restricted to 5 μg/g by the Commission E ofthe former Federal German Health Authority. The preloaded columns of theinvention are a useful tool for quantification of these toxic compounds.

Example 6. Quantifications of Biogenic Amines

Histamine is a biogenic amine, formed during the fermentation process inbeverage production like wine. It is vasoactive and can affect bloodpressure in higher concentrations as well as cause headaches,allergy-like reactions such as hives (urticaria), and even severe foodpoisoning. Thus the quantification of histamine in beverages is a usefultool for quality control in alcoholic beverages.

Example 7. Identification of Prohibited Compounds

A broad application is to use preloaded pipette tips or SPE columns ofthe invention for the identification of prohibited compounds in bodyfluids. Examples are for instance PDE-5 inhibitors and α-blockers inherbal OTC products to treat erectile dysfunction or the identificationof prohibited preservatives in certified natural cosmetics. The tips orcolumns of the invention are pre-loaded with a single concentration ofthe standard, enabling internal calibration. The standard is prepared ina specified, known amount (above the limit of quantification (LOQ)) withknown signal response magnitudes. The technology may be used for HPLCretention-time and/or mass-spec based substance identification.

Example 8. Clinical Diagnostics

The combination of the high automation capability, the safe use and thebroad range of application make the preloaded application a useful toolfor clinical diagnostics, especially in combination of FDA approval.

Example 9. PFAS Analysis of Drinking Water

Samples are prepared and analyzed according to procedures described inEPA METHOD 533: Determination of Per- and Polyfluoroalkyl substances indrinking water by isotope dilution anion exchange solve phase extractionand liquid chromatography/tandem mass spectrometry.

However, rather than using analytical standards prepared as compoundsobtained from vials, the standards are provided as columns onto whichstandards are adsorbed. Samples are processed with columns with andwithout adsorbed standards and the analysis is performed by standardaddition analysis.

Similar methods can be used to determine PFAS in waste water or innatural and commercial products.

We claim:
 1. A method of quantitating PFAS in a sample, comprising:adsorbing PFAS in the sample to solid phase extraction media and PFASstandard compounds, wherein the PFAS standard compounds are adsorbed tosaid solid phase media, and wherein the solid phase extraction media isfree of elution reagent; eluting the PFAS and PFAS standard compoundsfrom the solid phase extraction media with an elution solvent; andanalyzing the eluted PFAS, wherein the PFAS standard compounds are usedas internal standards.
 2. The method of claim 1, wherein the eluting isperformed in a bidirectional mode.
 3. The method of claim 1, wherein theeluting is performed in a unidirectional mode.
 4. The method of claim 1,wherein the solid phase extraction media is an ion exchanger.
 5. Themethod of claim 1, wherein the solid phase extraction media ishydrophobic interaction, reverse phase or normal phase.
 6. The method ofclaim 1, wherein the solid phase extraction media is inorganic, organicor polymeric.